sign in sign up
<<
Home , Weaning

ÔØ ÅÒÙ×Ö ÔØ

Natural and abrupt involution of the affects differently the metabolic and health consequences of weaning

Nissim Silanikove

PII: S0024-3205(14)00291-4 DOI: doi: 10.1016/j.lfs.2014.02.034 Reference: LFS 13945

To appear in: Life Sciences

Received date: 22 December 2013 Revised date: 6 February 2014 Accepted date: 25 February 2014

Please cite this article as: Silanikove Nissim, Natural and abrupt involution of the mam- mary gland affects differently the metabolic and health consequences of weaning, Life Sciences (2014), doi: 10.1016/j.lfs.2014.02.034

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT

Natural and abrupt involution of the mammary gland affects differently the metabolic and health consequences of weaning

Nissim Silanikove

Affiliation: Biology of Lactation Lab.,

Institute of Animal Science,

Agricultural Research Organization (ARO)

PO 6, Bet Dagan 50 250, Israel

Tel: 972-8-9484436

Email: [email protected]

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

ABSTRACT

In most under natural conditions weaning is gradual. Weaning occurs after the mammary gland naturally produces much less milk than it did at peak and established lactation. Involution occurs following cessation of milk evacuation from the mammary glands. Abrupt termination of evacuation of milk from the mammary gland at peak and established lactation induces abrupt involution. Evidence on mice has shown that during abrupt involution, mammary gland utilize some of the same tissue remodeling programs that are activated during wound healing. These results led to the proposition of the "involution hypothesis". According to the involution hypothesis, involution is associated with increased risk for developing breast cancer. However, the involution hypothesis is challenged by the metabolic and immunological events that characterize the involution process that follows gradual weaning. It has been shown that gradual weaning is associated with pre-adaption to the forthcoming break between dam and offspring and is followed by an orderly reprogramming of the mammary gland tissue. As discussed herein, such response may actually protect the mammary glands against development of breast cancer and thus, may explain the protective effect of extended .

On the other hand, termination of breastfeeding during the first 6 month of lactation likely associated with abrupt involution and thus with increased risk for developing breast cancer. Review of the literature on the epidemiologyACCEPTED of breast cancer principally MANUSCRIPT supports those conclusions.

Keywords: lactation, mammary gland, weaning, involution, breastfeeding, breast cancer

ACCEPTED MANUSCRIPT

The Critical Question

Development of the mammary glands secretory tissue occurs mostly at the end of pregnancy (Capuco and Akers, 1999; Lund et al., 1996). In most freely behavior mammals, including humans (Neville et al.,

1991), mice (Quarrie et al., 1996) and ruminants (Capuco and Akers, 1999) involution occurs following cessation of suckling by the offspring’s. Involution is the process whereby redundant mammary epithelial cells (MEC) are removed, which is one of the most notable examples of physiologically regulated cell death in an adult tissue (Capuco and Akers, 1999; Lund et al., 1996). Usually, involution occurs at the declining part of lactation where suckling rate and milk secretion are at their lowest level and the offspring already partially consumes post-weaning food. Natural weaning (NW) is influenced by circumstantial events that occur along the lactation such as, disease of the offspring or dam, rate of non- milk food consumption, etc. Weaning is therefore usually scattered within extended time interval, even in a group of humans or animals that gave at about the same time. Conducting an experiment, however, requires similarity among test groups; particularly toward the assessed biological hypothesize.

To overcome this difficulty, involution is induced abruptly at the beginning of lactations. At this stage, individual differences between the dams are still small. The majority of the information about developmental, metabolic and immunological outcomes of involution in farm (Capuco and Akers, 1999) and experimental animalsACCEPTED (Faupel-Badger et a., 2013 MANUSCRIPT) are based on implying early-lactation-forced- involution (ELFI) procedures. ELFI procedures comprised: teat sealing (Lie et al., 1997), unilateral cessation of milking of single gland (Quarrie et al., 1994), abrupt removal of young’s, or complete cessation of milking (Shamay et al., 2002). However, there is no evidence to support the inherent assumption; namely, that outcome of ELFI procedures essentially reflects metabolic and immune result of NW. ACCEPTED MANUSCRIPT

The first stage of involution (stage I), which covers the events occurring between the induction of milk stasis and extensive degradation of the secretory tissues (Stage II), is associates with a widespread apoptosis of the alveolar epithelial cells (Lund et al., 1997). Evidence based on ELFI procedures in mice has shown that during involution the mammary gland (MG) utilizes tissue remodeling programs that are activated during wound healing (Clarkson et al., 2004; Stein et al., 2004). Specifically, amplification of set of gens typical to those aroused during wound healing at the very first phase of involution (12 h) was found to be associated with immune cell infiltration and short-term formation of wound healing matrix components (Clarkson et al., 2004; Stein et al., 2004). Because similar changes confer a risk for developing cancer, it was suggested that MG involution per se may represent a window of opportunity for tumor cell progression (Clarkson et al., 2004; Lyons et al., 2011; Maller et al., 2010; McDaniel et al.,

2006; O'Brien et al., 2010; Schedin et al., 2004; Stein et al., 2004). This theory was coined by a panel of experts that reviewed the relationships among postpartum remodeling, lactation, and breast cancer as the "involution hypothesis" (Faupel-Badger et al., 2013). Thus, the question whether MG involution induced by ELFI procedures resemble the process followed by NW becomes critically important, particularly, from human health perspective. Similar thought was expressed by the above-mentioned panel of experts (Faupel-Badger et al. ., 2013).

The challenging data ACCEPTED MANUSCRIPT

A recent study in dairy cows has shown that involution induced under conditions that resembled NW, behaved like an orderly tissue remodeling programs. The involution under NW-like conditions was associated with effective clearing of apoptotic cells and produce conditions for eradicating or preventing new bacterial infections (Silanikove et al., 2013). On the other hand, involution in cows under forced weaning (FW)-like situation resembled the involution induced by ELFI procedures in mice. As in mice,

FW-like involution was associated with distressed wound-healing like response (Silanikove et al., 2013). ACCEPTED MANUSCRIPT

The involution under FW-like conditions was associated with inferior ability to prevent bacterial invasion into the mammary gland. In the rest the overview, scientific literature is critically evaluated in order to substantiate the concept that involution induced under NW and FW are fundamentally different from each other.

What is the basic difference between natural and abrupt involution?

Mammary gland involution can be induced at each stage of lactation, even at the very beginning of lactation and during peak of milk yield, by inducing milk stasis. Unilateral cessation of milking in 's

(Quarrie et al., 1994), and teat sealing in mice (Lie et al., 1997) induced involution only in the treated gland. This specific response indicates that mammary involution is triggered by local milk-born stimuli.

Several proposals for explaining the mechanism behind the negative feed-back regulation of milk secretion were proposed (Pai et al., 2008; Qualino et al., 2009; Silanikove et al., 2005, 2006). However, it seems that much more remained to be delineated in order to understand the physiological basis that underlies this feed-back process.

Involution in NW-like cows was associated with metabolic and immunological adaptations toward the expected disconnection between dams and their offspring, which was interpreted as an auto-defense ACCEPTED MANUSCRIPT mechanism (Silanikove et al., 2013). The large scale of cell apoptosis that characterize MG involution stage I requires effective clearance of the dead cells to avoid necrosis and uncontrolled inflammation.

The metabolic and immunological adaptations in NW-like cows allowed MG involution to proceed as an orderly tissue remodeling programs (Silanikove et al., 2013).

The first conspicuous phenological feature of NW-like and FW-like involution is disruption of the tight junctions (TJ) between MEC (Silanikove et al., 2013). When mammary gland involution was induced in ACCEPTED MANUSCRIPT

nonadapted (FW) cows, the response was associated with nuetrophilia and lymphopanea and resembled the general response to wounding (silanikove et al., 2013). The large influx of neutrophils to the MG was regarded as a response to urgent situation. The neutrophils serves as ‘gate keepers’ of the open TJ in order to prevent life threatening bactermia (penetration of bacteria from the gland lumen to the blood system) and penetration of milk proteins such as, casein and casein derived peptides

(McFadden et al., 1988), which can induce uncontrolled inflammation (sepsis) in the blood (Metcalf et.,

1996; Silanikove et al., 2013).

In NW-like involution total disruption of the TJ was preceded by partial disruption of the TJ. The partial opening of the TJ is important for developing auto-defense responses. The pre-involution opening of the

TJ in NW cows was associated with inflammatory response. However, the inflammatory response in NW- like cows was distinctively different from the acute response observed in cows undergoing FW-like involution: it was characterized by a balance presentation among neutrophils, T-cells (CD4 and CD8 subtypes) and macrophages in the gland lumen (Silanikove et al., 2013). The above-described response most likely prevents the stress caused by sudden total disruption of the TJ.

To which extent the findings in non-human species are relevant to human? ACCEPTED MANUSCRIPT Mice serve as the prevailing experimental model for evaluating biological questions important for human where direct experiments are not possible, or ethical (e.g., Vanhooren and Libert, 2013). Mice are small, cheap and have short reproductive cycle and life span, which makes them convenient model for testing biological concepts. However, models on inflammation and immune response based on experiment with mice were shown time and again not to precisely predict behavior in human beings

(Cauwelsa et al., 2013; Osterburg et al., 2013; Seok et al., 2013). Experimental results from given mammalian species may be used to build useful conceptual models relevant to different species, if it ACCEPTED MANUSCRIPT

help generate testable hypotheses for ultimate verification in the target species. From this viewpoint, it is considered useful to exploit the extensive information on the biology of lactation in farm animals.

Some of the biological features of farm animals, such as pregnancy and lactation cycle length and rate of involution in cows are more similar to human than those of mice. In comparison to the mammary glands of mice and rats, which can resorb milk components entirely and complete involution within approximately 7 days (Lund et al., 1997), the involuting gland of women remained functional for a long period, with secretion still being present in the lumen of the MG for up to 42 days after the abrupt termination of breast feeding (Hartmann et al., 1978). The involution process in FW-like cows is of about the same duration as found in humans (Shamay et al., 2003; Silanikove et al., 2005).

In the rest of the overview, the scientific literature on MG involution in different species is critically reviewed in order to derive testable hypotheses.

NW and FW are dissimilar in mice as well

Natural weaning in mice occurred when the young become accustomed to solid food (Shipman et al.,

1987). Involution in NW mice proceeds without any evidence of alveolar distension caused by milk stasis and is reflected by an orderly reprogramming involution process (Quarrie et al., 1994). On the other hand, FW is characterizedACCEPTED in mice by milk accumulation MANUSCRIPT and encroachment of the MG (Quarrie et al., 1994). These distinctive differences between NW and FW in mice were not taken into consideration in deriving the "involution hypothesis".

Extensive disruption of the TJ between the epithelial cell of the alveoli mark the start of involution

As argued above, opening of the TJ is a key event that starts the involution process and whether it occurs in adapted or non-adapted animals is the major cause for the distinctive differences between NW ACCEPTED MANUSCRIPT

and FW. However, opening of the TJ at the start of involution was not considered in some key reviews of the process (Monks et al., 2009; Radisky et al., 2009; Stein et al., 2007; Watson et al., 2012) or in constructing the "involution hypothesis". However, there are clear-cut evidence that disruption of the TJ is the first phenological event of involution and represent an interspecies general phenomena, as found in human (Hartmann et al., 1978; Neville et al., 1991), mice (Pai et al., 2008; Quaglino et al., 2009;

Valloroski et al., 2000), (Ben Chedly et al., 2009; Shamay et al., 2002), and cows (Silanikove et al.,

2005, 2013). Furthermore, evidence from goats and cows showed that maintenance the TJ open is critical for transfer from reversal opening of the TJ to irreversible course of involution (Shamay et al.,

2002, 2003). In accord, evidence from mice indicated that the anatomical and biochemical changes associated with disruption of the TJ play a key role in mediating intracellular signal transductions associated with profound apoptosis in those cells (Quaglino et al., 2009; Valloroski et al., 2000). Thus, there is unambiguous evidence that sustain the view that disruption of the TJ between the epithelial cells composing the alveoli mark and rule the start of involution.

NW is preceded by adaptive period

As argued above, it was shown in cows that partial disruption of TJ in the period preceding the expected timing of NW provided adaptation toward the sudden and complete opening of the TJ. Similar ACCEPTED MANUSCRIPT adaptation were also found in sheep and goats (Leitner et al., 2011). In Mice, at day 15 of lactation a sharp drop in milk yield naturally occurs concomitantly with the pups consuming dry food (Shipman et al., 1987). Replacement of the natural litter with younger pups prevented the normal fall in milk secretion. However, several markers of differentiation decreased during the induced extended lactation (Shipman et al., 1987), which supports the concept of pre- weaning adaptation. In women, significant decrease in concentrations of milk protein and lactose and significant increase in the concentrations of chloride, and sodium were observed only when milk volume ACCEPTED MANUSCRIPT

fell below 0.4 L/d (Neville et al., 1991), compared with yield of 0.8 L/d to 2L/d in single and twin-bearing mothers and up to 3 L/d in woman nursing triples at peak and established lactation (Neville et al., 1991;

Saint et al., 1986). The natural changes occurring in lactating woman during the period preceding weaning were described as 'gradual weaning' (Neville et al., 1991) and are distinctively different from those occurring when weaning occurred abruptly. The main difference between 'gradual weaning ' and abrupt weaning is the sharper (from 20 mM to 80 mM) increase in Na concentration mammary secretion in the abrupt weaned infants (Hartmann and Kulski, 1978), which is a clear indication of disrupted TJ (Neville et al., 1991; Shamay et al., 2003). Thus, the concept that across 's adaptation toward expected weaning is associated with partial disruption of TJ between the alveolar epithelial cells is a rational postulation.

The orderly reprogramming process characterizing NW explain the protective effect of birth order against breast cancer

According to the "involution hypothesis" it is expected that higher birth order would be associated with higher risk to develop breast cancer because women who gave birth 3 times, or more are extra exposed to the cancer promoting effects of involution in comparison to those that gave no birth or only to one or ACCEPTED MANUSCRIPT two children. However, meta-analysis of epidemiological data has shown that higher birth order is inversely associated with breast cancer risk among breast-fed women (Beral et al., 1997; Nichols et al.,

2008). Meta-analysis of large data-set revealed a 4.3% reduction in risk of invasive breast cancer for each lactation order (Nichols et al., 2008). Women who had never breast-fed had a 1.4-fold increased risk of breast cancer. The protecting effect of lactation was particularly pronounced in women with 3 or more youngsters compared with first-or second-born women, or those who never gave birth (Nichols et al., 2008). ACCEPTED MANUSCRIPT

The susceptibility to breast cancer increases under conditions that reduce mammary epithelial differentiation, increase the expression of genes regulating cell proliferation, and down-regulate genes that improve DNA damage repair or induce apoptosis or differentiation (Henson and Tarone, 1994; Pike et al., 1983). Milk production is associated with imposition of oxidative stress on the MG (Silanikove et al., 2005, 2009, 2012), and oxidative stress considered for being one of the causes for the declining phase of lactation in mammals (Hadsell et al., 2007). Involution stage II of the mammary gland is associated with complete degradation of the MG parenchyma (lobular, alveolar tissues and the surrounding basal membrane). The vast majority of breast cancer originates from these structures

(Henson and Tarone, 1994; Pike et al., 1983). Thus, involution stage II allow the replacement of older, oxidatively damaged tissues with new tissue, which is free of oxidative injure toward the next pregnancy-lactation cycle. The smooth transition from involution Stage I to stage II in NW mammals in comparison to the delay in the process associated with exposing MG tissue to extra-oxidative stress in

FW mammals may explain the protective effect of NW against breast cancer. Multiparous women tent to breastfed their young for longer period and thus to terminate their lactation with NW (see discussion below).

Abrupt weaning may increase the risk of mothers to develop breast cancer: An explanation to the protective effect of longACCEPTED duration of breastfeeding MANUSCRIPT

The World Health Organization recommendations are that children will be breast-fed for their first 2 years of life (WHO, 2003), and those of the American Academy of Pediatrics (AAP, 2005) and the

American Academy of Family Physicians (AAFP, 2001) recommend exclusive breastfeeding for the first 6 months of life. However, those recommendations are far from being adopted in the general population in Western countries: In the United States, breastfeeding rates have increased significantly among infants born between 1993-1994 and 2005-2006 and 2007-2013. In 2013, the rates of breastfeeding was ACCEPTED MANUSCRIPT

high at 77%. However, of infants born in 2010, only 49% were breastfeeding at 6 months, up from 35% in 2000. The breastfeeding rate at 12 months in 2010 was 27%, up from 16% in 2000 (CDCP, 2013).

Breastfeeding duration was identified in many countries as protective against development of breast cancer: Germany (Chang-Claude et al., 2000); India (Gajalakshmi et al., 2009); Iran (Akbari et al., 2011);

Israel (Shema et al., 2007); Japan (Nagata et al., 2012); Tunis (Awatef et al., 2010) and Turkey (Newman,

2010). The above reports are in agreement with earlier large collaborative reanalysis of data from epidemiological studies comprising 52,705 women with breast cancer and 108,411 women without breast cancer (Beral et al., 1997). It was concluded that every 12 months of breastfeeding decrease the risk of breast cancer by 7.0% (p<0.0001), in addition to the protective contribution of birth order. It was estimated that the cumulative incidence of breast cancer in developed countries would be reduced by more than half; from 63 to 27 per 100,000 women by age 70, if women had the average number of and lifetime duration of breast-feeding that had been prevalent in developing countries.

Breastfeeding accounted in this analysis for almost two-thirds of the estimated reduction in breast cancer incidence (Beral et al., 1997).

In general, women from industrialized western societies, face a notably elevated lifetime risk of developing breast cancerACCEPTED (Fig 1), which range with MANUSCRIPT age-adjusted incidence rates from in excess of 100 per 100,000 population in the USA (Horner et al., 2009) to 20 per 100,000 population in East Africa (Fig

1). The rate of developing breast cancer in USA women is particularly high (Horner et al., 2009).

However, within the USA population the risk for developing breast cancer is significantly different among the ethnic populations; the numbers per 100,000 women being: White, 127.8; Black, 117.7;

Asian/Pacific Islanders, 89.5; American Indians/ Alaskan Native, 74.4; Hispanic, 83.3. The higher incidence of breast cancer found in developed countries was related to shorter duration of breastfeeding (or lack thereof), and was associated with lower parity. On the other hand, larger family ACCEPTED MANUSCRIPT

sizes and longer lifetime duration of breastfeeding in developing countries were estimated to cut the risk of breast cancer by one-half of that in developed countries (Beral et al., 1997). In addition to the information reviewed by Beral et al. (1997), additional published research further sustain the conclusion that long breastfeeding reduce the risk of acquiring breast cancer.

In the face of more than 50 incidents of breast cancer cases per 100,000 in the general population in

South America (Fig.1), virtually no breast cancer events were detected in survey of two rural indigenous populations in Brazil (Da Silva et al., 2008; Geimba et al., 2001). Women in those two populations are characterized by multiparty and much extended duration (> 3 years) of breastfeeding. Delay of first birth together with low parity and short duration of breast feeding reflect an increased social trends in countries where extended breastfeeding is traditional. For instance, in Korea about half of the children are traditionally breastfed for or as long as 3 years (Kim et al., 2007). Time-depended trend in the increase of breast cancer is particularly evident in countries in which there is a switch from traditional long-term breastfeeding to western countries style (Keinan-Boker et al., 2013; Moore et al., 2010; Shin et al., 2010). Recent findings on the association between breastfeeding and breast cancer in Korea have shown that breast cancer risk decreased according to the total months of breast-feeding (P for trend = 0.03). Average durationACCEPTED of breastfeeding of 11-12 MANUSCRIPTmonths reduced risk of breast cancer by as much as 54% compared with the duration of 1-4 months (Kim et al., 2007). In Sri Lanka, compared to 0–11 months of lifetime breast-feeding, there was a 66.3% reduction in breast cancer risk in women who breastfed for 12–23 months, 87.4% reduction in 24–35 months and 94% reduction in 36–47 months categories. The mean duration of breast-feeding per child for ≥12 months was also associated with reduced risk of breast cancer (De Silva et al., 2011).

In conclusion, the protective effect of extended breastfeeding against acquiring of breast cancer has profound physiological basis. Termination of breastfeeding between a few days after birth and up to 6 ACCEPTED MANUSCRIPT

month after is likely associate with involution that is induced abruptly. Abrupt involution is characterized by acute wounding-like response and therefore may be associated with increased risk for developing cancer. On the other hand, extended breastfeeding ends with NW. Involution that follows NW is characterized by an ordered reprogramming of the mammary tissue. This type of involution ends with completes or near complete degradation of the old parenchyma. As discussed above, such response may protect the MG against development of breast cancer and thus explain the protective effect of extended breastfeeding.

References

AAFP (American Academy of Family Physicians), 2007. Breastfeeding (Position Paper), 2001, 26/2/

2007. Available from:

Http://www.aafp.org/online/en/home/policy/policies/b/breastfeedingpositionpaper.html

AAP (American Academy of Pediatrics), 2005. Breastfeeding and the use of human milk. Pediatrics

115, 496–506.

Akbari, A., Razzaghi, Z.,ACCEPTED Homaee, F., Khayamzadeh, MANUSCRIPT M., Movahedi, M., Akbari, M.E., 2011. Parity and breastfeeding are preventive measures against breast cancer in Iranian women. Breast Canc. 18, 51-55

Awatef, M., Olfa, G., Imed, H., Kacem, M., Imen, C., Rim, C.,Mohamed, B ., Slim, B.A., 2010.

Breastfeeding reduces breast cancer risk: a case-control study in Tunisia. Canc. Cases Control

21, 393-397. ACCEPTED MANUSCRIPT

Ben Chedly, H.B., Lacasse, P., Marnet, P.-G., Wiart-Letort, S., Finot, L., Boutinaud, M., 2009. Cell junction

disruption after 36 h milk accumulation was associated with changes in mammary secretory

tissue activity and dynamics in lactating dairy goats. J. Physiol. Pharmacol. 60 (Suppl. 3), 105-

111.

Beral, V., Doll, R., Hermon, C., Peto, R., Reeves, G., 1997. Breast cancer and hormone replacement

therapy: collaborative reanalysis of data from epidemiological studies of 52,705 women with

breast cancer and 108,411 women without breast cancer. Lancet 350, 1047 – 1059.

Capuco, A.V., Akers, R.M. 1999. Mammary involution in dairy animals. J. Mammary Gland Biol. Neoplasia

4,137–144.

Cauwelsa, A., Vandendriessche, B., Brouckaert, P., 2013. Of mice, men, and inflammation. Proc. Nation.

Acad. of Sci. U.S.A. 110, E3150 (2013).

CDCP (Centers for Disease Control and Prevention), 2013. Breastfeeding report card 2013. Available from: http://www.cdc.gov/breastfeeding/data/NIS_data/index.htm.

Chang-Claude, J., Eby, N., Kiechle, M., Bastert, G., Becher, H., Breastfeeding and breast cancer risk by age

50 among womenACCEPTED in Germany. 2000. Canc. MANUSCRIPT Causes Control 11, 687-695.

Clarkson, R.W.E., Wayland, M.T., Lee, J., Freeman, T., Watson, C.J., 2004. Gene expression profiling of

mammary gland development reveals putative roles for death receptors and immune mediators

in post-lactational regression. Breast Canc. Res. 6, R92-R109. ACCEPTED MANUSCRIPT

Da Silva, E. P., Pelloso, S.M., Carvalho, M.D.D., Toledo, M.J.D., 2009. Exploring breast cancer risk factors

in Kaingang women in the Faxinal Indigenous Territory, Parana State, Brazil, 2008. Cadernos de

Saude Publica 25, 1493-1500.

De Silva, M., Senarath, U., Gunatilake, M., Lokuhetty, D., 2010. Prolonged breastfeeding reduces risk of

breast cancer in Sri Lankan women: A case-control study. Canc. Epidemiol. 34, 267 – 273.

Faupel-Badger, J.M., Jessica M., Arcaro, K. F., Balkam, J.J., Eliassen, A.H., Hassiotou, F., Lebrilla, C.B.,

Michels, K.B., Palmer, J.R. et al., 2013. Postpartum remodeling, lactation, and breast cancer

risk: Summary of a National Cancer Institute–sponsored workshop. J. Natl. Cancer Inst. 105,166–

174.

Gajalakshmi, V., Mathew, A., Brennan, P., Rajan, B., Kanimozhi, V.C., Mathews, A., Mathew, B.S.,

Boffetta, P., 2009. Breastfeeding and breast cancer risk in India: A multicenter case-control

study. Int. J. Canc. 125, 662-665.

Geimba de Lima, M., Koifman, S., Scapulatempo, I.L., Peixoto, M., Naomi, S., Curado do Amaral, M.,

2001. [Risk factors for breast cancer among rural Terena]. Cadernos de saude publica 17, 1537-

1544. ACCEPTED MANUSCRIPT Hadsell, D., George, J., Torres, D., 2007. The Declining Phase of Lactation: Peripheral or Central,

Programmed or Pathological? J. Mammary Gland Biol. Neoplasia 12, 59 – 70.

Hartmann, P.E., Kulski, J.K., 1978. Changes in the composition of the mammary secretion of women

after abrupt termination of breast feeding. J. Physiol. 275, 1-11 (1978).

Henson, D.E., Tarone, R.E., 1994. Involution and the Etiology of Breast Cancer. Canc. 74, 424-429. ACCEPTED MANUSCRIPT

Horner, M.J.R.L., Krapcho, M., Neyman, N., Aminou, R., Howlader, N., Altekruse, S.F., Feuer, E.J., Huang,

L., Mariotto, A., Miller, B.A., Lewis, D.R., Eisner, M.P., Stinchcomb, D.G., Edwards, B.K. (eds),

2009. SEER cancer statistics review, 1975–2006, based on November 2008 SEER data

submission, posted to the SEER web site. http://seer.cancer.gov/csr/1975_2006/. National

Cancer Institute, Bethesda

Keinan-Boker, L., Baron-Epel, O., Fishler, Y., Liphshitz, I., Barchana, M., Dichtiar, R., Goodman, M., 2013.

Breast cancer trends in Israeli Jewish and Arab Women, 1996-2007. Europ. J. Canc. Prev. 22,

112-120.

Kim, Y., Choi, J.-Y., Lee, K.-M., Park, S.K., Ahn, S.-H., Noh, D.-Y., Hong, Y.-C., Kang, D., Yoo, K.-Y., 2007.

Dose-dependent protective effect of breast-feeding against breast cancer among ever-lactated

women in Korea. Europ. J. Canc. Prev. 16, 124 – 129.

Leitner, G., Merin, U., Silanikove, N., 2011. Effect of glandular infection and stage of lactation on milk

clotting parameters: Comparison among cows, goats and sheep. Int. Dairy J. 21, 279 - 285.

Li, M., Liu, X.V.V, Robinson, G., BarPeled, U., Wagner, K.U., Young, W.S., Hennighausen, L., Furth, P.A.,

1997. Mammary-derived signals activate programmed cell death during the first stage of

mammary glandACCEPTED involution. Proc. Nation . MANUSCRIPTAcad. Sci., U. S. A. 94, 3425–3430.

Lund, L.R., Romer, J., Thomasset, N., Solberg, H., Pyke, C., Bissell, M.J., Werb, Z., 1996. Two distinct

phases of apoptosis in mammary gland involution: Proteinase-independent and dependent

pathways. Develop. 122,181–193.

Lyons, T.R., O'Brien, J., Borges, V.F., Conklin, M.W., Keely, P.J., Eliceiri, K.W., Eliceiri, Kevin W., Tan, A.-C.,

Schedin, P., 2011. Postpartum mammary gland involution drives progression of ductal

carcinoma in situ through collagen and COX-2. Nat. Med. 17, 1109–1115 (2011). ACCEPTED MANUSCRIPT

Maller, O., Martinson, H., Schedin, P., 2010. Extracellular matrix composition reveals complex and

dynamic stromal-epithelial interactions in the mammary gland. J Mammary Gland Biol

Neoplasia. 2, 301–318.

McDaniel, S.M., Rumer, K.K., Biroc, S.L., Metz, R.P., Singh, M., Porter, W., Schedin, P., 2006. Remodeling

of the mammary microenvironment after lactation promotes breast tumor cell metastasis. Am.

J. Pathol. 168, 608–620.

McFadden, T.B., Akers, R.M., Cappuco, A.V., 1988. Relationships of milk proteins in blood with somatic

cell counts in milk of dairy cows. J. Dairy Sci. 71, 826–834.

Metcalf, D., Robb, L., Dunn, A.R., Mifsud, S., DiRago, L., 1996. Role of granulocyte-macrophage colony-

stimulating factor and granulocyte colony-stimulating factor in the development of an acute

neutrophil inflammatory response in mice. Blood 88, 3755-3764.

Monks, J., Henson, P.M., 2009. Differentiation of the mammary epithelial cell during involution:

Implications for breast cancer. J. Mammary Gland Biol. Neoplasia 14, 159–170.

Moore, M.A., Eser, S., Igisinov, N., Igisinov, S., Mohagheghi, M.A., Mousavi-Jarrahi, A., Ozenturk, G., Soipova, M., Tuncer,ACCEPTED M., Sobue, T., 2010. CancerMANUSCRIPT Epidemiology in South Asia - past, present and future. Asian Pacific J. Canc. Prev. 11, 49 – 66.

Nagata, C., Mizoue, T., Tanaka, K.,Tsuji, I., Tamakoshi, A., Wakai, K., Matsuo, K., Ito, H., Sasazuki, S.,

2012. Breastfeeding and breast cancer risk: An evaluation based on a Systematic Review of

epidemiologic evidence among the Japanese population. Jap. J. Clin. Oncol. 42, 124-130. ACCEPTED MANUSCRIPT

Neville, M.C., Allen, J.C., Archer, P.C., Casey, C.E., Seacat, J., Keller, R.P., Lutes, V., Rasbach, J., Neifert,

M., 1991. Studies in Human lactation – milk volume and nutrient composition during weaning

and lactogenesis. Amer. J. Clin. Nutr. 54, 81-92.

Newman, L.A., 2010. Applicability of the Gail model for breast cancer risk assessment in Turkish female

population and evaluation of breastfeeding as a risk factor. Breast Canc. Res. Treat. 120, 425-

426.

Nichols, H.B., Trentham-Dietz, A., Sprague, B., Hampton, J.M., Titus-Ernstoff, L., Newcom, P.A., 2008.

Effects of birth order and maternal age on breast cancer risk. Epidemiology 19, 417-423.

O’Brien, J., Lyons, T., Monks, J., Lucia, M.S., Wilson, R.S., Hines, L., Man, Y.-G., Borges, V., Schedin, P.,

2010. Alternatively activated macrophages and collagen remodeling characterize the

postpartum involuting mammary gland across species. Am. J. Pathol. 176, 1241–1255.

Osterburg, A.R., Andrew R., Hexley, P., Supp, D.M., Robinson, C.T., Noel, G., Ogle, C., Boyce, S.T.,

Aronow, B.J., Babcock, G.F., 2013. Concerns over interspecies transcriptional comparisons in

mice and humans after trauma. Proc. Nation. Acad. of Sci. U.S.A. 110, E3370.

Pai, V.P., Horseman, NACCEPTED.D., 2008. Biphasic Regulation MANUSCRIPT of Mammary Epithelial Resistance by

Serotonin through Activation of Multiple Pathways. J. Biol. Chem. 283, 30901–30910 (2008).

Pike, M.C., Krailo, M.D., Henderson, B.E., Casagrande, J.T., Hoel, D.G., 1983. “Hormonal” risk factors,

“breast tissue age” and the age-incidence of breast cancer. Nature 303: 767-70.

Quarrie, L.H, Addey, C.V.P., Wilde, C.J., 1994. Local regulation of mammary apoptosis in the lactating

goat. Biochem. Soc. Transactions 22, 178S. ACCEPTED MANUSCRIPT

Quarrie, L.H, Addey, C.V.P., Wilde, C.J., 1996. Programmed cell death during mammary involution

induced by weaning, litter removal and milk stasis J. Cell. Physiol. 168, 559–569.

Quaglino, A., Salierno, M., Pellegrotti, J., Rubinstein, N., Kordon, E.C., 2009. Mechanical strain induces

involution-associated events in mammary epithelial cells. BMC Cell Biol. 10, 55.

Radisky, D.C., Hartmann, L.C., 2009. Mammary involution and breast cancer risk: Transgenic models and

clinical studies. J. Mammary Gland Biol. Neoplasia 14, 181–191.

Saint, L., Maggiore, P., Hartmann, P.E., 1986. Yield and nutrient content of milk in 8 breast-feeding twins

and one are breast-feeding triplets. Br. J. Nutr. 56, 49-58.

Schedin, P., Mitrenga, T., McDaniel, S., Kaeck, M., 2004. Mammary ECM composition and function are

altered by reproductive state. Mol. Carcinog. 41, 207–220.

Seok J., Junhee; W.H., Shaw; C., Alex G., Mindrinos, M., Baker, H.V., Xu, W.H., Richards, D.R., McDonald-

Smith, G.P., et al., 2013. Genomic responses in models poorly mimic human

inflammatory diseases. Proc. Nation. Acad. of Sci. U.S.A. 110, 3507–3512.

Shamay, A., Shapiro, F., Mabjeesh, M.J., Silanikove, N., 2002. Casein-derived phosphopeptides disrupt

tight junction integrity,ACCEPTED and precipitously dryMANUSCRIPT up milk secretion in goats. Life Sci. 70, 2707–2719.

Shamay, A., Shapiro, F., Leitner, G., Silanikove, N., 2003. Infusions of casein hydrolyzates into the

mammary gland disrupt tight junction integrity and induce involution in cows. J. Dairy Sci. 86,

1250–1258.

Shema, L., Ore, L., Ben-Shachar, M., Haj, M., Linn, S., 2007. The association between breastfeeding and

breast cancer occurrence among Israeli Jewish women: a case control study. J. Canc. Res. Clin.

Oncol. 133, 539-546. ACCEPTED MANUSCRIPT

Shin, H.-R., Joubert, C., Boniol, M., Hery, C., Ahn, S.H.,Won, Y.-J.,Nishino, Y., Sobue, T., Chen, C.-J. et al.,

2010. Recent trends and patterns in breast cancer incidence among Eastern and Southeastern

Asian women. Canc. Cases Control 21, 1777-1785.

Shipman, L.J., Docherty, A.H., Knight, C.H., Wilde, C.J., 1987. Metabolic adaptations in mouse mammary

gland during normal lactation cycle and in extended lactation. Quart. J. Exp. Physiol. 72, 303-

311.

Silanikove, N., Shapiro, F., Shamay, A., Leitner, G., 2005. Role of xanthine oxidase, lactoperoxidase and

NO in the innate immune system of mammary secretion during active involution in dairy cows:

Manipulation with casein hydrolysates. Free Radic. Biol. Med. 38, 1139–1151.

Silanikove, N., Merin, U., Leitner, G., 2006. Physiological role of indigenous milk enzymes: An overview

of an evolving picture. Int. Dairy J. 16, 533–545.

Silanikove, N., Shapiro, F., Silanikove, M., Merin, U., Leitner, G., 2009. Hydrogen peroxide-dependent

conversion of nitrite to nitrate as a crucial feature of bovine milk catalase. J. Agric. Chem. Food

Sci. 57, 8018 – 8025.

Silanikove, N., Rauch-Cohen,ACCEPTED A., Shapiro, F., Arieli, MANUSCRIPT A., Merin, U., Leitner, G., 2012. Lipopolysaccharide challenge of the mammary gland in cows induces nitrosative stress that impairs milk oxidative

stability. Anim. 6, 1451-1459.

Silanikove, N., Merin, U., Shapiro, F., Leitner, G., 2013. Early mammary gland metabolic and immune

responses during natural-like and forceful drying-off in high-yielding dairy cows. J. Dairy Sci. 96,

6400–6411. ACCEPTED MANUSCRIPT

Stein T., Morris, J.S., Davies, C.R., Weber-Hall, S.J., Duffy, M.A., Heath, V.J., Bell, A.K., Ferrier, R.K.,

Sandilands, G.P., Gusterson, B.A., 2004. Involution of the mouse mammary gland is associated

with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3. Breast

Canc. Res. 6, R75–R91.

Stein, T., Salomonis, N., Gusterson, B.A., 2007. Mammary gland involution as a multi-step process. J.

Mammary Gland Biol. Neoplasia 12, 25–35.

Vanhooren, V., Libert, C., 2013. The mouse as a model organism in aging research: Usefulness, pitfalls

and possibilities. Ageing Res. Rev. 12, 8-21.

Vallorosi, C. J., Day, K.C., Zhao, X., Rashid, M.G., Rubin, M.A., Johnson, K.R., Wheelock, M.J., Day, M.L.,

2000. Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis

during prostate and mammary involution J. Biol. Chem. 275, 3328–3334.

Watson, C.J., Kreuzaler, P.A., 2012. Remodeling mechanisms of the mammary gland during involution.

Int. J. Dev. Biol. 55, 757-762.

WHO (World Health Organization), 2003. Global Strategy for Infant and Young Child Feeding, Geneva.

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

Figure legend

Breast cancer: Incidence and death in different parts of the world. Source: Breast cancer research, UK. http://www.cancerresearchuk.org/cancer-info/cancerstats/world/

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

Natural weaning Abrupt weaning

ACCEPTED MANUSCRIPT 1

Graphical abstract